Cardiac contraction in a healthy human heart is initiated by spontaneous excitation of the sinoatrial (“SA”) node located in the right atrium. The electric impulse generated by the SA node travels to the atrioventricular (“AV”) node where it is transmitted to the bundle of His and to the Purkinje network. The fibers in the Purkinje network branch out in many directions to facilitate coordinated contraction of the left and right ventricles. In some disease states, the heart loses some of its natural capacity to pace properly. Such dysfunction is commonly treated by implanting an electronic pacemaker.
While effectively improving the lives of many patients, implantable pacemakers have certain limitations. For example, implantable pacemakers rely on a self-contained power source such as a battery and consequently have a limited lifetime before the power source is in need of replacement. Hence, an otherwise healthy patient may require multiple surgeries to replace the power source or the entire implantable pacemaker. Also, implantable pacemakers may not directly respond to physiological signals similar to the way the SA node responds to such signals. Furthermore, there is the risk of infections associated with implantable pacemakers, which, while infrequent, can be catastrophic. Also, there is the potential for interference from other devices.
These problems could be solved with the creation of biological (i.e., cell-based) cardiac pacemakers that would be naturally integrated into or created within the heart with deficient pacemaker function. While many cell types and gene targeting strategies have been suggested, the progress has been limited. Biological methods of influencing a patient's cardiac cells have been developed, some of which include administering biopharmaceutical compositions that affect cardiac pacing. Developments in genetic engineering have produced methods to make pacemaker-like cells from non-pacemaker cardiac cells or to regenerate the pacing capabilities of cells in the conduction system of the heart. For example, U.S. Pat. No. 6,214,620 describes a method for modulating the excitability of ventricular cells by controlling the regulation of the expression of certain ion channels (e.g. K+ channels). WO 02/087419 and WO 05/062890 describe methods and systems for modulating electric behavior of cardiac cells by genetic modification of inwardly rectifying K+ channels (IK1) in quiescent ventricular cells.
Another biological approach for moderating cardiac pacing involves implanting into the SA node or other suitable heart regions cells having hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. For example, see WO 02/098286 and WO 05/062958A2. It is disclosed that, physiologically originating in the SA node, the HCN channels play a role in the control of rhythmic electrical heart activity. Cyclic nucleotides modulate the HCN channel activity, and channel activation occurs upon hyperpolarization rather than depolarization, generating an inward current, called “funny” current (If). There are four isoforms of HCN channels (HCN1-4), and each has greater or lesser prevalence in different heart regions. Because the HCN isoforms, generating the If current, are believed to be directly involved in generation and modulation of diastolic depolarization of cardiac pacemaker cells, implantation of HCN-expressing cells into cardiac tissue that is diseased or experiencing conduction blockage has been suggested as a viable method for regulating cardiac pacemaker function.
One group demonstrated that by injection of the adenylate cyclase VI gene into the heart ventricular muscle a biological cardiac pacemaker could be created (Ruhparwar A, et al., 2007, Scientific Sessions of the American Heart Association, Orlando, Fla.). Adenylyl cyclase 6 (AC6) is expressed in ventricular cells and is inhibited by Ca2+. According to Ruhparwar et al., cyclic AMP (cAMP) is generated in response to β-adrenergic receptor (β-AR) stimulation and also binds to HCN channels, where it regulates spontaneous rhythmic activity in the heart. But targeting HCN channels cannot generate “funny” current (If) in adult ventricular myocytes because these cells do not express HCN channels and generate only a negligible If current. There are two more major problems with this approach: absence of sustainability and absence of rhythmicity of spontaneous excitations. Previous studies showed that fast pacing in the presence of β-AR stimulation can indeed activate Ca2+ cycling in ventricular myocytes, but this spontaneous activity is not sustained; it is only temporary, just a few beats, and cannot be rhythmic because it is associated with arrhythmogenic full-cell-length Ca2+ waves. The Ca2+ waves in ventricular or atrial myocytes are known to produce cardiac arrhythmia and therefore cannot provide a pacemaker function. Indeed, while each Ca2+ wave generates its Delayed After-Depolarization (DAD), not every DAD results in the cell excitation. Spontaneous Ca2+ waves and their DAD-induced excitations (if any) in cells with over-expression of β-ARs or Ca2+ inhibitable AC6 do not occur under normal conditions but require extrinsic stimulation: electrical (fast pacing), nerve (to produce catecholamines), chemical, and/or hormonal (to activate β-ARs). Indeed, Ruhparwar et al. used rapid ventricular pacing combined with administration of isoprenaline (to activate β-ARs). The strong dependence on the extrinsic factors in cells with the Ca2+ inhibited AC6 is not surprising, because the cAMP production is inhibited (and therefore not sustained) when the sarcoplasmic reticulum (SR) releases Ca2+.
Another approach is to use in vitro-grown embryonic stem (ES) cell-derived cardiac cells (ESCs) as biological pacemakers. Since ESCs generate spontaneous action potentials (APs) and functionally integrate with ventricular myocytes in vitro and with the host mycocardium in vivo, they can pace hearts after their implantation (as reported in guinea pigs and pigs) and even overcome complete atrioventricular block in animal models. While these results show that these cells can potentially function as biological pacemakers, there are still some fundamental problems remaining with this approach.
One major problem is that ESCs are highly heterogeneous cells; they express major cardiac ion currents and Ca2+ cycling proteins, but many ESCs generate irregular APs that could be proarrhythmic and, therefore, cannot be used in humans. Currently, there is no effective means to identify and select so called “sinus-node-like” cells generating rhythmic APs other than to directly measure those APs and/or chronotropic responses to neuromediators. While previous studies have suggested that several factors contributed to ESC automaticity, as shown in FIG. 1, such as interplay of Ca2+ and K+ currents, classical If mechanism and ICaT, INa window current, global Ca2+ oscillations activating a nonselective cation current, and different types of local Ca2+ releases (LCRs) from SR, their complex interactions and roles to generate spontaneous Aps are unknown. The requirements for ESCs to generate rhythmic APs also remain unknown.
The complex nature of cardiac pacemaker function and regulation has been the subject of debate and intensive experimental studies. It has been widely believed that the If current, sometimes called “the pacemaker current”, is the dominant mechanism of cardiac pacemaker function and the heart rate regulation. More recent studies in genetically manipulated mice demonstrated, however, that the cardiac pacemaker function and heart response to β-AR stimulation are preserved in the absence of If or in the absence of cAMP sensitivity of If. Alternatively, based on previous studies in cardiac Purkinje cells, pacemaker oscillations were described to be the resultant of a surface membrane oscillator and a subcellular rhythm generator, which is largely independent from the surface membrane. The strongly coupled function of Ca2+ and membrane oscillators/clocks in the heart's natural pacemaker, sinoatrial node is now emerging as a novel fundamental principle of cardiac impulse initiation and regulation (Lakatta E G, et al., 2010 Circ Res 106: 659-673).
What is needed are biological pacemakers engineered to intrinsically generate excitations which, similar to those of natural pacemakers, must be rhythmic and sustained for many years, in the best case for the rest of the patient's life.